The invention relates to therapy for treating neoplasia, particularly cancer.
In America, half of all men and one-third of all women will develop some form of neoplasia during their lifetimes. For example, millions of Americans are currently living with cancer or have been cured of the disease. Although cancer is frequently fatal, rapid treatment of the disease results in a better prognosis for recovery.
Neoplasia is caused by the abnormal growth and proliferation of cells in the body. While normal body cells grow, divide, and die in an orderly fashion, neoplastic cells grow and divide in a disorderly fashion. Such disorderly growth during neoplasia may be caused by abnormal dysregulation of the cell cycle leading to the hyper-proliferation of cells (Sherr (1996) Science 274: 1672-1677).
In eukaryotic cells, progression through the cell cycle is orchestrated by functionally distinct cydin-dependent kinases (CDK""s). Complexes are formed via the association of different catalytic CDK domains and regulatory cyclin subunits. CDK4/cydin D and CDK6/cyclin D complexes regulate progression through G1 phase; CDK2/cyclin E kinase regulates the G1/S transition; CDK2/cyclin A complex drives the cells through S-phase; and CDC2/cyclin B complex controls the entry, and exit from mitosis (Sherr (1996) Science 274: 1672-1677). The activity of the CDKs is tightly regulated in response to a variety of extra- and intracellular signals, and is mediated through a combination of phosphorylation events and associations with cyclin-dependent kinase inhibitors (CDKi""s) (Morgan (1995) Nature 374: 131-134). The redistribution of CDKi""s between the different CDK/cyclin complexes during the cell cycle coordinates the timing of activation and de-activation of the kinase activities (Sherr and Roberts (1995) Genes and Dev. 9: 1149-1163).
Two classes of structurally distinct CDKi""s, the CIP/KIP and the INK4 families, have been identified in mammalian cells. The CIP/KIP family includes p21/CIP1/WAF1, p27/KIP1, and p57/KIP2 (Sherr and Roberts, supra). CIP/KIP family members share a conserved N-terminal region of an inhibitory domain having a length of approximately 60 amino acids. The members of the CIP/KIP family are potent inhibitors of the CDK4, CDK2, and CDC2 kinases, although their over-expression in vivo predominantly leads to arrest in the G1 phase of the cell cycle. The members of the INK4 family include p15/INK4a, p16/INK4b, p18/INK4c, and p19/INK4d (Serrano et al. (1993) Nature 366: 704-707; Hannon and Beach (1994) Nature 371: 257-261; Hirai et al. (1995) Mol. Cell. Biol. 15: 2672-2681; Chan et al. (1995) Mol. Cell. Biol. 15: 2682-2688; Guan et al. (1996) Mol. Biol. Cell. 7: 57-70). The INK4 proteins are comprised almost exclusively of the repetitions of a common structural motif, the ankyrin repeat (Bork (1993) Proteins 17: 363-374), and are highly specific inhibitors of the CDK4 and CDK6 associated kinases.
Biochemical analysis of the members of the CIP/KIP and INK4 families of inhibitors has suggested that their modes of action are different. p16, the prototypic member of the INK4 family, binds to both monomeric CDK4 and to the assembled, fully active CDK4/cyclin D1 kinase. The binding of p16 to CDK4 appears to prevent the formation of CDK4/cyclin D complexes, whereas binding of p16 to assembled CDK4/cyclin D complex produces a catalytically inactive ternary complex (Parry et al. (1995) EMBO J. 14: 503-511). p27 binds primarily to the active CDK2/cydinA and CDK2/cyclinE complexes, which leads to the formation of a catalytically inactive ternary complex (Polyak et al. (1994) Cell 78:1156-66; Polyak et al. (1994) Genes and Dev. 8: 9-22).
To inhibit cell cycle progression, the levels of the individual CDKi""s must exceed the concentration of the active CDK/cyclin complexes. Control of cell proliferation has been shown to act through the regulation of the levels of various CDKi""s. For example, under growth conditions that induce arrest of cell proliferation, the amount of p27 increases (Firpo et al. (1994) Mol. Cell. Biol. 14: 4889-4901; Kato et al. (1994) Cell 79: 487-496; Nourse et al. (1994) Nature 372: 570-573, Slingerland et al. (1994) Mol. Cell. Biol. 14: 3683-3694). As the cells are stimulated to reenter the cell cycle, the level of p27 is promptly reduced. Similarly, as cells become senescent, p16 gradually increases (Alcorta et al. (1996) Proc. Natl. Acad. Sci. USA 93: 13742-13747).
Given the large numbers of human deaths attributable to neoplasia, there is a need to develop useful methods and compositions to treat or prevent neoplasia, including hyperplasia and cancer. Moreover, given the important role that the cell cycle plays in several disease phenotypes, reagents that arrest cell growth are useful in allowing further study of the cell cycle for synchronizing and controlling growth of cells in culture.
The present features novel chimeric cyclin dependent kinase inhibitors (chimeric CDKi""s) which, when combined with an adenovirus E4 protein (or active fragment thereof), not only inhibit neoplastic cell growth, but surprisingly also induce apoptosis in neoplastic cells. Using the chimeric CDKi protein, W9, apoptosis was induced in neoplastic cells regardless of their p53, Rb, p27, or p16 status. The combination of a chimeric CDKi with an adenovirus E4 protein does not induce apoptosis in most normal cells, but rather only induces cell growth arrest. These surprising discoveries have been exploited to provide the anti-neoplastic compositions and methods of the present invention.
Accordingly, in a first aspect, the invention features a nucleic acid composition consisting essentially of a first nucleic acid sequence encoding a chimeric CDKi protein and a second nucleic acid sequence encoding an adenovirus E4 protein, wherein the first and second nucleic acid sequences are operably linked to at least one regulatory sequence. Such nudeic add sequences are capable of being expressed in a cell. In certain embodiments, the chimeric CDKi protein is a W9 protein. In certain embodiments, the adenovirus E4 protein is encoded by E4orf6.
In another aspect, the invention features a composition comprising the chimeric CDKi protein/adenovirus E4 protein-encoding nudeic acid composition according to the first aspect of the invention and a pharmaceutically acceptable carrier. In one embodiment, the composition further comprises a delivery system that facilitates the internalization of the composition by a cell. Preferably, the delivery system is a recombinant virus particle. In other embodiments, the recombinant virus particle is selected from the group consisting of an adenovirus, a lentivirus, an adeno-associated virus, a retrovirus, a herpesvirus, and a vaccinia virus. Preferably, the recombinant virus particle is an adenovirus (e.g., an adenovirus lacking an entire E4 region). In another embodiment, the delivery system is a liposome.
In yet another aspect, the invention features a nucleic acid composition comprising a first nucleic acid sequence encoding a secretable, intemalizable form of a chimeric CDKi protein and a second nudeic acid sequence encoding a secretable, internalizable form of an adenovirus E4 protein, wherein the first and second nudeic acid sequences are operably linked to at least one regulatory sequence. In certain embodiments, the chimeric CDKi protein is a W9 protein. In certain embodiments, the adenovirus E4 protein is encoded by E4orf6.
In another aspect, the invention features a composition comprising a nucleic acid composition comprising a first nucleic acid sequence encoding a secretable, internalizable form of a chimeric CDKi protein and a second nucleic acid sequence encoding a secretable, internalizable form of an adenovirus E4 protein, and a pharmaceutically acceptable carrier, wherein the first and second nucleic acid sequences are operably linked to at least one regulatory sequence. In certain embodiments, the composition further comprises a delivery system that facilitates the internalization of the composition by a cell. Preferably, the delivery system is a recombinant virus particle. In other embodiments, the recombinant virus particle is selected from the group consisting of an adenovirus, a lentivirus, an adeno-associated virus, a retrovirus, a herpesvirus, and a vaccinia virus. Preferably, the recombinant virus particle is an adenovirus. In another embodiment, the delivery system is a liposome.
In another aspect, the invention features a protein composition comprising a purified chimeric CDKi protein and a purified adenovirus E4 protein. In certain embodiments, the chimeric CDKi protein is a W9 protein. In certain embodiments, the adenovirus E4 protein is encoded by E4orf6.
In yet another aspect, the invention features a composition comprising a protein composition comprising a purified chimeric CDKi protein and a purified adenovirus E4 protein, and a pharmaceutically acceptable carrier. In one embodiment, the composition further comprises a delivery system that facilitates the internalization of the composition by a cell.
In still another aspect, the invention features a protein composition comprising a purified, internalizable form of a chimeric CDKi protein and a purified, internalizable form of an adenovirus E4 protein. In certain embodiments, the chimeric CDKi protein is a W9 protein. In certain embodiments, the adenovirus E4 protein is encoded by E4orf6.
In another aspect, the invention features a composition comprising a protein composition comprising a purified, internalizable form of a chimeric CDKi protein and a purified, internalizable form of an adenovirus E4 protein, and a pharmaceutically acceptable carrier.
In yet another aspect, the invention features a method for treating an animal with a neoplasm. The method includes administering to the animal a therapeutically effective amount of a composition according to the invention.
In another aspect, the invention features a cell containing a nucleic acid composition comprising a first nucleic acid sequence encoding a secretable, internalizable form of a chimeric CDKi protein and a second nucleic acid sequence encoding a secretable, internalizable form of an adenovirus E4 protein, wherein the first and second nucleic acid sequences are operably linked to at least one regulatory sequence. In certain embodiments, the chimeric CDKi protein is a W9 protein. In certain embodiments, the adenovirus E4 protein is encoded by E4orf6.
In yet another aspect, the invention features a method for treating an animal with a neoplasm, comprising introducing the nucleic acid composition comprising a first nucleic acid sequence encoding a secretable, internalizable form of a chimeric CDKi protein and a second nucleic acid sequence encoding a secretable, internalizable form of an adenovirus E4 protein, wherein the first and second nucleic acid sequences are operably linked to at least one regulatory sequence into a cell of the animal, wherein the introduced cell secretes the secretable, internalizable form of the chimeric CDKi protein and secretes the secretable, internalizable form of the adenovirus E4 protein.
In final aspect, the invention features a method for treating an animal with a neoplasm, comprising introducing the nucleic acid composition comprising a first nucleic acid sequence encoding a secretable, internalizable form of a W9 protein and a second nucleic acid sequence encoding a secretable, internalizable form of an adenovirus E4 protein, wherein the first and second nudeic acid sequences are operably linked to at least one regulatory sequence into a cell of the animal, wherein the introduced cell secretes the secretable, intemalizable form of the W9 protein and secretes the secretable, internalizable form of the adenovirus E4 protein.